CN112725280B - Thiodimethylarsinic acid induced human keratinocyte malignant transformation cell strain and application thereof - Google Patents
Thiodimethylarsinic acid induced human keratinocyte malignant transformation cell strain and application thereof Download PDFInfo
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Abstract
The invention discloses a malignant transformation cell strain of human keratinocyte caused by thio-dimethyl arsonic acid and application thereof, belonging to the technical field of model construction. The invention constructs an inorganic arsenic metabolite, namely the sulfo-dimethyl arsonic acid (DMMTA), by continuously infecting and culturing human keratinocytes with the sulfo-dimethyl arsonic acidV) The malignant transformation cell model of the human keratinocyte helps to identify the carcinogenicity of the arsenic methylation metabolite, and indicates that the arsenic metabolite is sulfodimethylarsinic acid (DMMTA)V) The low-dose long-term exposure can cause malignant transformation of skin cells, and provides a new cell model basis and a new research idea for the research of arsenic carcinogenesis mechanisms.
Description
Technical Field
The invention relates to a malignant transformation cell strain of human keratinocyte caused by thio-dimethyl arsonic acid and application thereof, belonging to the technical field of model construction.
Background
Inorganic arsenic and its compounds are determined carcinogens in human, and the metabolism of inorganic arsenic in the body is often considered as a detoxification process in the past, however, the research in recent years shows that the inorganic arsenic generates a high-toxicity product of sulfodimethylarsinic acid (DMMTA) in the metabolism processV) This may be closely related to the development of cancer. However, the lack of relevant animal models has not revealed the specific mechanism of carcinogenesis, which results in the lack of effective early diagnosis and treatment in clinical practice.
At present, the research of the carcinogenic mechanism of arsenic mainly uses an in vitro cell malignant transformation model constructed by chronic contamination of inorganic arsenic, and the cell malignant transformation model is a material for better researching the carcinogenic mechanism of carcinogenic substances. However, the in vitro cell malignant transformation model constructed by chronic inorganic arsenic infection has limitations, and the metabolic transformation of arsenic in vivo and the high toxicity of metabolites are not fully considered. In addition, as the genome of human cells is relatively stable and has an effective DNA repair mechanism, the human cells have stronger resistance to external damage, especially damage to carcinogens is not as sensitive as some animal cells, and transformation is relatively difficult compared with animal cells, no transformation model of the thio-dimethylarsinic acid cells is reported at present.
Disclosure of Invention
In order to solve the problems, the invention constructs a model of malignant transformation cells of human keratinocytes caused by an inorganic arsenic metabolite, namely thio-dimethyl arsonic acid, is beneficial to identifying and identifying carcinogenicity of arsenic methylated metabolites, and also provides a new thought and model basis for research of an arsenic carcinogenic mechanism.
The invention aims to provide a malignant transformation cell strain of human keratinocyte caused by sulfodimethylarsinic acid, which is preserved in China general microbiological culture Collection center (CGMCC) within 12 months and 30 days in 2020, with the preservation number of CGMCC No.21419 and the preservation address of No. 3 Hospital No.1 of Beijing, Chaoyang, and the cell strain is prepared by carrying out cell culture transformation on human keratinocyte caused by sulfodimethylarsinic acid.
The second purpose of the invention is to provide a method for transforming and constructing a malignant transformation cell strain of human keratinocyte caused by thio-dimethyl arsonic acid, which specifically comprises the steps of carrying out continuous contamination culture on human keratinocyte by adopting a culture medium containing 0.5-1.0 mu M of thio-dimethyl arsonic acid, changing liquid every 20-30 h, carrying out subculture for expansion when the cell fusion degree reaches 75-85%, and carrying out subculture to 30-40 generations to obtain the malignant transformation cell strain of human keratinocyte caused by thio-dimethyl arsonic acid.
Further, the sulfodimethylarsinic acid is prepared by the following method:
s1, dissolving dimethylarsinic acid and sodium sulfate in water, slowly adding concentrated sulfuric acid, and stirring and uniformly mixing for 10-24 hours, wherein the molar ratio of the dimethylarsinic acid to the sodium sulfate to the concentrated sulfuric acid is 1: 1.5-2;
s2, adding a hydrochloric acid solution into the mixed solution after stirring for dehydration reaction, and extracting by using chloroform after the reaction;
s3, adding saturated saline solution into the organic phase obtained in the step S2, and collecting the lower clear liquid;
s4, adding anhydrous calcium chloride into the lower clear liquid, collecting the upper clear liquid, and heating to remove water to obtain a solid;
and S5, recrystallizing the solid obtained in the step S4 by using hexane, and removing the hexane to obtain the sulfodimethylarsinic acid.
Further, the recrystallization is performed for 10-20 hours at the temperature of 0-4 ℃.
Further, the culture medium is a DMEM high-sugar complete culture medium.
Further, the DMEM high-glucose complete medium contains 80-120 mug/ml streptomycin, 80-120U/ml penicillin and 8-12% fetal calf serum.
Furthermore, the application also comprises the detection of MMP-9 secretion, cell migration or soft agar clone formation of cells, which indicates that the cells are subjected to malignant transformation.
The third purpose of the invention is to provide the application of the malignant transformation cell strain of the human keratinocyte caused by the sulfodimethylarsinic acid in a cell model for researching the carcinogenic mechanism of the sulfodimethylarsinic acid.
The fourth purpose of the invention is to provide the application of the malignant transformation cell strain of human keratinocyte caused by the sulfodimethylarsinic acid in screening or evaluating the medicines for treating cancers caused by inorganic arsenic.
The invention has the beneficial effects that:
the invention constructs an inorganic arsenic metabolite sulfodimethylarsinic acid (DMMTA)V) The malignant transformation cell model of the human keratinocyte helps to identify the carcinogenicity of the arsenic methylation metabolite, and indicates that the arsenic metabolite is sulfodimethylarsinic acid (DMMTA)V) The low-dose long-term exposure can cause malignant transformation of skin cells, and provides a new cell model basis and a new research idea for the research of arsenic carcinogenesis mechanisms.
Biological material preservation:
the human keratinocyte malignant transformation cell strain caused by the thio-dimethyl arsonic acid is preserved in China general microbiological culture Collection center (CGMCC) at 03.06.2020, with the preservation number of CGMCC No.19650 and the preservation address of No. 3 Hosiebola No.1 of Beijing Kogyo Chen.
Drawings
FIG. 1 shows DMMTA at various concentrations (0-2. mu.M)VCell survival 24 hours after treatment of HaCaT cells;
FIG. 2 is a 1.0 μ M DMMTAVContinuously infecting the change of MMP-9 secretion of cells after different generations of HaCaT cells; note: *: p < 0.05 compared to passage control (no treatment);
FIG. 3 is a 1.0 μ M DMMTAVThe change of cell doubling time after different generations of HaCaT cells are continuously infected with virus; note: *: p < 0.05 compared to passage control (no treatment);
FIG. 4 is a 1.0 μ M DMMTAVChange of cell migration capacity after 35 generations of continuously infected HaCaT cells; note: the left image is an observation image under a scratch experiment microscope of the 35 th generation of infected and control group cells; the right panel is a quantitative analysis of the left panel; *: p < 0.05 compared to passage control (no treatment);
FIG. 5 is a 1.0 μ M DMMTAVChange of colony forming ability of cell clone after 35 generations of continuously infected HaCaT cell; note: the upper graph is an observation image under a soft agar clone experiment microscope of 35 generation of infected group and control group cells and a quantitative analysis result image thereof; *: p was < 0.05 compared to the passage control (no treatment).
Detailed Description
The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.
Example 1:
inorganic arsenic metabolite Thiodimethyl arsonic acid (DMMTA)V) The synthesis of (2): taking DMA by balanceV(molecular weight 138) powder 5g (about 35.6mmol) and sodium sulfide (Na)2S) 4.5g (about 57.0mmol) of the powder was dissolved in 150mL of ultrapure water. Then concentrated sulfur is addedAcid was slowly added to the mixture stepwise (final molar ratio H)2SO4:Na2S:DMAV1.6:1.6:1), the mixture was stirred overnight under argon using a magnetic stirrer. Adding a hydrochloric acid solution to the mixture the next day to make DMMTA in the mixtureVDehydration reaction occurs, and then organic solvent chloroform is added for extraction. Next, saturated saline solution was further added, and the lower clear solution was collected in a beaker. Anhydrous calcium chloride was added to the collected supernatant, and the supernatant was collected and then heated in a water bath to remove water. Followed by the addition of the organic solvent Hexane and traces of DMMTAVThe solid was recrystallized and placed in a 4 ° freezer overnight. The mixture was taken out of the refrigerator every other day and further added with an organic solvent, Hexane, and then evaporated to dryness under an argon atmosphere to obtain a white solid. And finally, verifying the obtained white solid by using an analysis method combining high performance liquid chromatography and inductively coupled plasma mass spectrometry (HPLC-ICP-MS).
Determining the appropriate infectious dose of DMMTA at different concentrations (0-2. mu.M)VAfter 24 hours of HaCaT cell treatment, the survival of the cells was examined by the CCK8 method, and the dose with the highest cell survival rate was used as the long-term toxicant dose, as shown in FIG. 1, DMMTAVThe long-term exposure dose of (A) was determined to be 1.0. mu.M.
Example 2:
2 dishes of normal HaCaT cells (O generation cells) were prepared, and one dish was filled with 1.0. mu.M DMMTAVThe DMEM high-glucose complete culture medium (containing 100 mu g/ml streptomycin, 100U/ml penicillin and 10% fetal calf serum) is continuously subjected to contamination culture for a long time, cell liquid is changed every 24 hours, subculture is carried out when the cell fusion degree reaches 80%, and the subculture is carried out to 35 generations. And the other dish is normally cultured by a DMEM high-glucose complete medium (containing 100 mu g/ml streptomycin, 100U/ml penicillin and 10% fetal calf serum), cell liquid is changed every 24 hours, subculture is carried out when the cell fusion degree reaches 80%, and the cell liquid is subjected to subculture to 35 generations as a subculture control group. The doubling time of the 0 th, 1 st, 7 th, 14 th, 21 th, 28 th and 35 th generation cells of the infected group and the control group was measured by setting a monitoring point every 7 th generation.
Example 3:
passing through the inspectionThe matrix metalloproteinase-9 (MMP-9), cell doubling time, cell migration ability and the change of independent growth of cell anchorage are measured to identify whether the cells are subjected to malignant transformation. FIG. 2 reflects the secretion change of MMP-9 from different generations of HaCaT cells, from which it can be seen that the secretion of MMP-9 is significantly increased when cultured to 21, 28, and 35 generations under contamination, compared with the passage control group. FIG. 3 reflects the variation of doubling time of HaCaT cells in different generations, from which it can be seen that the doubling time of the cells is significantly shortened up to 28 and 35 generations in the infected culture. FIG. 4 is a scratch test for detecting the change of migration ability of HaCaT cells after 35 generations of contamination culture, and it can be seen that the migration ability of cells after 35 generations of contamination culture is significantly enhanced. FIG. 5 is a soft agar cloning experiment for observing the change of independent growth anchored by cells when the cells are cultured to 35 generations, and it can be seen that the cells of the passage control group only form individual micro-colonies in the soft agar, while the cells of the 35 generations infected with virus can form obvious cloning colonies in the soft agar, and the cloning formation rate is obviously higher than that of the cells of the passage control group. The secretion of MMP-9, cell doubling time, cell migration ability and soft agar colony forming ability of the cells are common indexes for identifying malignant transformation of cells in vitro. Based on the above experimental results, long term exposure to 1.0 μ M Thiodimethyl arsonic acid (DMMTA) was demonstratedV) And the HaCaT cells can be induced to generate malignant transformation when subcultured to 35 generations.
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.
Claims (3)
1. A malignant transformed cell strain of human keratinocyte caused by thioanisole arsonic acid is preserved in China general microbiological culture Collection center (CGMCC) within 30 months of 2020, with the preservation number of CGMCC No.21419 and the preservation address of No. 3 Hospital No.1 of Beijing, Chaoyang, respectively.
2. Use of the malignant transformed cell line of human keratinocyte derived from thioanisolide arsonate as claimed in claim 1 in a cell model for studying the carcinogenic mechanism of thioanisolide arsonate.
3. Use of the malignant transformed cell line of human keratinocyte of thioanisolide arsonate as claimed in claim 1 for screening or evaluating drugs for treating cancer caused by inorganic arsenic.
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| PCT/CN2021/077088 WO2022156027A1 (en) | 2021-01-25 | 2021-02-20 | Cell strain for malignant transformation of human keratinocytes caused by thiodimethylarsenic acid and use thereof |
| US17/631,478 US20230159910A1 (en) | 2021-01-25 | 2021-02-20 | Dimethylmonothioarsinic acid-induced malignantly transformed cell line of human keratinocytes and use thereof |
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